Desmosome dualism: most of the junction is stable but a plakophilin moiety is persistently dynamic

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes – desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from calcium-dependence to calcium-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome down-regulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes.

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Desmosomal dualism: the core is stable while plakophilin is dynamic

10.1101/2021.03.02.433631 ◽

2021 ◽

Author(s):

Judith B Fülle ◽

Henri Huppert ◽

David Liebl ◽

Jaron Liu ◽

Rogerio Alves de Almeida ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Core Stability ◽

Cell Junctions ◽

Major Protein ◽

Desmosomal Cadherins ◽

The Core ◽

Cell Scattering ◽

Stable Core ◽

Protein Classes

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calciumindependent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.

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Live cell imaging shows hepatocyte growth factor-induced Met dimerization

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2016.04.015 ◽

2016 ◽

Vol 1863 (7) ◽

pp. 1552-1558 ◽

Cited By ~ 7

Author(s):

David Koschut ◽

Ludovic Richert ◽

Giuseppina Pace ◽

Hartmut H. Niemann ◽

Yves Mély ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Hepatocyte Growth

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Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration

The Journal of Cell Biology ◽

10.1083/jcb.201007003 ◽

2011 ◽

Vol 193 (1) ◽

pp. 61-70 ◽

Cited By ~ 97

Author(s):

Zhizhan Gu ◽

Erika H. Noss ◽

Victor W. Hsu ◽

Michael B. Brenner

Keyword(s):

Functional Analysis ◽

Cell Migration ◽

Growth Factor ◽

Live Cell Imaging ◽

Cell Imaging ◽

Focal Adhesions ◽

Initial Step ◽

Ventral Surface ◽

Platelet Derived Growth Factor ◽

Trailing Edges

During cell migration, integrins are redistributed from focal adhesions undergoing disassembly at the cell’s trailing edges to new focal adhesions assembling at leading edges. The initial step of integrin redistribution is thought to require clathrin-mediated endocytosis. However, whether clathrin-mediated endocytosis functions in different contexts, such as basal versus stimulated migration, has not been determined. In this paper, we examine the spatial and temporal redistribution of integrins from focal adhesions upon stimulation by growth factors. Four-dimensional confocal live-cell imaging along with functional analysis reveals that surface integrins do not undergo significant endocytosis at ventral focal adhesions upon cell stimulation with the platelet-derived growth factor. Rather, they abruptly redistribute to dorsal circular ruffles, where they are internalized through macropinocytosis. The internalized integrins then transit through recycling endosomal compartments to repopulate newly formed focal adhesions on the ventral surface. These findings explain why integrins have long been observed to redistribute through both surface-based and internal routes and identify a new function for macropinocytosis during growth factor–induced cell migration.

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Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2185 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2185-2200 ◽

Cited By ~ 233

Author(s):

Sandra Potempa ◽

Anne J. Ridley

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Map Kinase ◽

Adherens Junctions ◽

Adherens Junction ◽

Dominant Negative ◽

Cell Junctions ◽

Scatter Factor ◽

Cell Scattering ◽

Hepatocyte Growth

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.

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Quantification of growth factor signaling and pathway cross talk by live-cell imaging

AJP Cell Physiology ◽

10.1152/ajpcell.00312.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. C328-C340 ◽

Cited By ~ 6

Author(s):

Sean M. Gross ◽

Peter Rotwein

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Signaling Pathways ◽

Cross Talk ◽

Live Cell Imaging ◽

Cell Imaging ◽

Akt Signaling ◽

Erk Signaling ◽

Signaling Cascades

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor–receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras–Raf–Mek–ERK and phosphatidylinositol (PI) 3-kinase–Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways.

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High-throughput formation and image-based analysis of basal-in mammary organoids in 384-well plates

Scientific Reports ◽

10.1038/s41598-021-03739-1 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Soojung Lee ◽

Jonathan Chang ◽

Sung-Min Kang ◽

Eric Parigoris ◽

Ji-Hoon Lee ◽

...

Keyword(s):

Growth Factor ◽

High Throughput ◽

Live Cell Imaging ◽

Cell Imaging ◽

Automated Analysis ◽

Time Lapse ◽

Culture Conditions ◽

Live Cell ◽

Future Research ◽

Heparin Binding

AbstractThis manuscript describes a new method for forming basal-in MCF10A organoids using commercial 384-well ultra-low attachment (ULA) microplates and the development of associated live-cell imaging and automated analysis protocols. The use of a commercial 384-well ULA platform makes this method more broadly accessible than previously reported hanging drop systems and enables in-incubator automated imaging. Therefore, time points can be captured on a more frequent basis to improve tracking of early organoid formation and growth. However, one major challenge of live-cell imaging in multi-well plates is the rapid accumulation of large numbers of images. In this paper, an automated MATLAB script to handle the increased image load is developed. This analysis protocol utilizes morphological image processing to identify cellular structures within each image and quantify their circularity and size. Using this script, time-lapse images of aggregating and non-aggregating culture conditions are analyzed to profile early changes in size and circularity. Moreover, this high-throughput platform is applied to widely screen concentration combinations of Matrigel and epidermal growth factor (EGF) or heparin-binding EGF-like growth factor (HB-EGF) for their impact on organoid formation. These results can serve as a practical resource, guiding future research with basal-in MCF10A organoids.

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